EXPERIENTIA - USP · 2015-03-17 · plant is, in its first phase, a straightforward one. C02 , 1 ~o2 h~ l LEAF T H~ % T (CH20) Fig. 2. We may visualize the problem in terms of the

EXPERIENTIA Vol. VII I - Fase. 12 Pag. 445-484 15. XII . 1952

The Path of Carbon in Photosynthesis

(XX. The Steady State)

By M. CALVIN and P. MASSlNI 2, Berkeley, Cal.

Photosynthesis, the process by which green plants are able to capture electromagnetic energyfin the form of sunlight and transform it into stored chemical energy in the form of a wide variety of reduced (relative to carbon dioxide) carbon compounds provides the only major source of energy for the maintenance and pro- pagation of all life. For this and other reasons, the study of the nature of this process has been a very attractive area for many years and a wide variety of scientific interest and backgrounds have been brought to bear upon it. These range from the purely biological to the strictly physical with the biochemical and phy- sicochemical area lying between. Important contri- butions to the understanding of the phenomenon have come from all these areas, but in spite of the enormous amount of work and study that has gone into the problem, relatively little is known, or rather understood, about the fundamental character of the process even today. I t is perhaps pardonable that one engaged in studies in this area tends'to the conclusion that most of the knowledge has been acquired in the relatively recent past. Discounting that tendency, it still seems fair to say that we have only just begun in the last decade or so to gain some understanding of the intimate details by which the basic process represented in the overall reaction

+ h v C02 + H20 ~ 02 + (CH20)~

<

- - E n e r g y

has come to be understood. The recognition of this overall reaction as written, to represent the basic nature of-the process of photosynthesis, and, further, that its reversal represents the basic reaction of respiration is, of course, an old one.

As a result of more recent study, it has been possible to separate the process of photosynthesis into two dis- tinct and separate parts. The gener.al features of this

1 The work described in this paper was sponsored by the U.S. Atomic Energy Commission.

2 Radiat ion Laboratory and Depar tment of Chemistry, Univer- s i ty of California, Berkeley. Fellow of the Swiss Foundation, ,,Stiftung ffir Stipendien auf dem Gebiete tier Chemie~,, 1951-1952.

separation may be represented in the following chart (Fig. 1). The essential feature of the separation is the independence of the photochemical part of photosynthesis from the carbon dioxide reduction part. We shall not here even t ry to outline all of the various forms of evidence Which have been adduced in support of such a scheme but only to point out additional bits which have been added in recent years and particularly those which stem from our own work'.

(CH20) 0

C02

L • 2 0

, " "9 . " _j

Fig. 1.

Tile scheme itself is an outgrowth of proposals of some fifteen years ago by VAN NIEL 2 resulting from his studies of the comparative biochemistry of photosynthesis. More recently, the photochemical apparatus has been shown to be separable from the rest of the plant by the experiments of Hill 3.

He was able to make prepardtions of chloroplasts and chloroplastic fragments which, upon illumination in the presence of suitable oxidizing agents other than carbon dioxide, were able to evolve molecular oxygen. Still more recently, OCHOA an others 4 were able to demonstrate that these same preparations were capable of using coenzyme I and II (D.P.N. and T.P.N.) as

x M. CALVIN and A. A. BENSON, Science 107, 476 (1948). - A. A. BENSON and M. CALVtN, Cold Spring Harbor Symp. quant. Biol. 13, 6 (1948). - M. CAt.VlN and A. A. BENSON, Science 10.9, 140 (1949).

2 C, B, VAN NIEL, Photosynthesis in Plants, Chapter 22 (Iowa State College Press, Ames, Iowa, 19.19), pp. -t37--195.

a R. HILL, Nature 139, 881 (19.17); Prec. roy. See. (London) [B] 127, 192 (1939).- R. HZLL and R. SeARISBRICK, Nature 146, 61 (19,t0).

4 W. VISHtCIAC and S. Octm*, J. Biol. Chem. 196, 75 (1952). - D. I. ARrCON, Nature 167, 1008 (1951). - I.. J. TOLMAClt, Arch. Bio- chem. Biophys. 83, 120 (1951).

446 M. CALVIn and P. MASSINI: The Path of Carbon in Photosynthesis [ExPERIENTIAVOI..VIII/12]

suitable oxidizing agents leading to the evolution of oxygen. Furthermore, the experiments of RUBE~ 1 showed that the molecule of oxygen evolved in photosynthesis had its proximate origin in the oxygen of the water molecule and that the oxygen atom associ- ated with the carbon dioxide must first pass through water before arriving at gaseous oxygen. From the chart it may be seen that the ultimate result, then, of the photochemical reaction initiated by the absorption of light by the chlorophyll molecule is the division of the water molecule into an oxidized part which ultimately leads to molecular oxygen and some reduced parts represented in the chart by [H t.

This reduced part [H l we have called "reducing power" because as yet it is not possible to state specifi- cally what form or forms it may be in. This reducing power is capable of reducing carbon dioxide in the absence of light; that is to say, that the reduction of carbon dioxide itself is a dark reaction. This was indicated first in the earlier experiment of McALISTER ~ in which he was able to show that following a period of photosynthesis a number of plants continued to absorb carbon dioxide for a short period (seconds to minutes) after cessation of illumination. We were able to demonstrate this in an even more direct and unequivocal fashion and generalize it for all plants so far tried when we were able to show that not only did all of these plants absorb quantities of carbon dioxide in the dark after illumination but that the products formed in the dark were qualitatively and under certain conditions quantitatively similar to those formed in a fairly com- parable light period s . The method used for this demonstration was the same as those to be described later in the review. The lifetime in the dark of this reducing power which is generated by light is also of the order of seconds to minutes and almost certainly corresponds to a concentration of one or more definite chemical species. I t is quite conceivable, as mentioned earlier, that some of it might be in the form of reduced coen- zymes.

Very recently it has been reported 4 that both the higher plants and isolated chloroplasts emit a chemiluminiscence following cessation of illumination. This chemiluminiscence has a decay time which corresponds very closely to that which we have observed for the reducing power. In fact, it would seem almost surely to represent the reversal of the conversion of electromagnetic into chemical energy, namely, the transformation of at least some of the chemical energy stored in the reducing power into the electromagnetic energy of luminiscence. Furthermore, the luminiscence is re-

1 S. RUBEN, M. RANDALL, M. D. KAMEN, and J. HYDE, J. Am. Chem. Soc. 63, 877 (1941).

2 E. D. MCALtsTER and J. MYERS, J. Smithsonian Inst. Pubt. (Misc. Coil.) 6, 99 (1940).

3 M. CALVIN, J. Chem. Education 26, 639 (1949). 4 B. L. STREHLER and W. ARNOLD, J. Gen. Physiol. 34, 809

(1951). - B. [.. ~TREHLER, Arch. Biochem. Biophys. 34, 289 (1951).

duced by the presence of carbon dioxide in those cases in whic.h the carbon dioxide fixing system is still present. However, when the carbon dioxide system has been removed, as is true in the case of chloroplasts, the luminiscence becomes independent of carbon dioxide.

While it thus appears tha t the unique problem of photosynthesis lies in the right hand half of the chart of Figure 1, the present discussion will be limited to the other side of the chart, that is, the path through which carbon passes on its way from carbon dioxide to all the raw materials of the plant. It is essentially a study of what we now believe to be entirely dark reactions and might best be characterized as phyto- synthesis. This area not only has a great interest for its own sake but would almost certainly cast some light upon the nature of the reducing agents which arrive from the photochemical part of the reaction and drive the carbon cycle toward reduction. The reason for this particular interest lies in the fact that we have, in recent years, come into possession of a tool which is especially suited for this study, namely, labeled carbon atoms in the form of a radioactive isotope of carbon, C 14. All of the results tha t will be described later were made possible through the use of this labeled carbon dioxide. With such a labeled molecule available, the design of an experiment for determining the sequence of compounds into which the carbon atoms of carbon dioxide may pass during the course of their incorporation in the plant is, in its first phase, a straightforward one.

C02 ,

1 ~o2

h~

l LEAF

T H~

%

T

(CH20)

Fig. 2.

We may visualize the problem in terms of the chart in Figure 2 in which the green leaf is represented schematically as a closed opaque container into which stream the raw materials of photosynthesis, namely, carbon dioxide, light and water containiiag the necessary mineral elements. From this container are evolved tile products of photosynthesis--oxygen gas and the reduced carbon compounds constituting the plant and its stored reserves. Heretofore, it has been possible to study in a quantitative way the nature of the process going on inside the opaque container only by varying external conditions and noting variations in the final products. Although there has been no serious doubt that the formation of sugar did not take place by the aggregation of six molecules of carbon dioxide, six

•15. XII. 195% M. C,~.LVlN and P, MAssIst: The Path of Carbon in Photosynthesis 447

molecules of water and the requisite number of light quanta into a single unit followed by the rearrangement into hexose and molecular oxygen, no specific infor- mation was available as to the compounds which might act as intermediates. Assuming that such a chain of intermediates exists, it is quite clear that by setting up some photosynthetic organism, leaf or other suitable material, in a steady state of photosynthesis in which the various ingredients are being absorbed and products formed in some uniform manner and injecting the labeled carbon dioxide into the entering carbon dioxide stream, we should find the label appearing successively in time in that chain of intermediates. This can be observed by stopping the entire process after a suitable lapse of time and examining the incorporated labeled carbon to determine the nature of the compounds into which it has been built. It is also clear that in addition to the identity and sequence of the compounds into which the carbon is incorporated, we may also determine the order in which the various carbon atoms within each compound acquire the label. With this type of infor- mation at hand it should be possible to reconstruct the sequence of events from the time of entry of the carbon atom into tlae plant as carbon dioxide until it appears in the various more or less finished products of the plant.

Fig. 3.--Algae Plant.

While photosynthetic experiments have been done with a vide variety of plant materials, the major kinetic work has been carried out with suspensions of unicellular green algae. The reason for this lies in the

Fig. 4.--"Lollipop".

fact that these algae may be obtained in a reproducible biological form relatively easily and in any amount. They are grown in the laboratory in a continuous cul- ture arrangement shown in Figure 3. The algae may be harvested from these flasks daily or every other day, depending upon the type of material desired. Such cultures have been maintained in a continuous fashion over periods extending beyond several months. Most of our experiments have been performed with the unicellular green algae Chlordla or Scenedesmus. After harvesting the algae are washed with distilled water and resuspended in the medium in which the experiment is to be done. This suspension is placed in a flat vessel called a "lollipop", a photograph of which is shown in Figure 4. A stream of air containing carbon dioxide is passed through the algae while they are being illuminated so as to achieve a steady state of photosynthesis.

In order to begin the experiment the air stream is interrupted and the labeled bicarbonate is injected into the algal suspension. After the preselected period of time, the algae are killed by opening the large stopcock at the bottom of the flask, allowing the algal suspension to fall into alcohol in order to stop the reaction and extract the photosynthesized material. Although a variety of killing and extracting procedures have been tested, most of the experiments were performed by dropping the algae into alcohol so as to result in an 80% alcohol solution. The total amount of carbon fixed is then determined by taking an aliquot of this entire suspension, evaporating it to dryness on an alu- minum disk and counting it on a GEmER counterL The fraction soluble is determined by either filtering or centrifuging the suspension and then recounting the clear supernate or filtrate.

The distribution of the fixed radiocarbon among the various compounds must now be determined. Since in

1 M. CALVIN, C. HEIDELBERGER, J. C. REID, B. M. TOLBERT, and P. E. YANKWICt[, Isolopid Carbon (John x, Viley & Sons, Inc., New York, 19 I9).

448 ~1. CALVIN and P. 31ASSINI : "l'tle Path of Carbon in Photosynthesis [EXI'ERIENTIA\'OL.VI I 1/12j

relatively short periods of t ime most of the fixed radio- 8.o act ivi ty is found in the soluble components, the prob- t s lem is one of analyzing for the distribution in the soluble fraction. This has been done by an application 72 of the method of paper chromatography introduced s.o and developed for amino acid analysis by CONSBEN,

6.4 MARTIN, and SYNGE 1. I t has since been applied to a - wide var iety of compounds and no detailed description ~ 6.o of it will be given here. The unique extension to our ~ s

~t work lies in the ability to locate particularly those [,5~ compounds which contain the radioactive carbon atoms o on the paper by means of a radioautograph of the ~,.6 resulting paper chromatogram obtained by allowing an 4, X-ray film to remain in contact with the paper for a

,640 suitable period of time. Those areas of the paper which ~ i

g 36~ are occupied by radioactive compounds will, of course, e expose the X-ray film. Such a map of the disposition ~ 32 of the radioactive compounds contained in an extract ~ z~ is shown in Figure 5. The chemical nature of the com- 2.4 pounds defined by the exposed areas can be inferred from the position occupied by a compound with re- z c spects to the origin of the chromatogram. More precise )e determination of the chemical character is assisted by ~z chemistry performed on the material eluted from the spot defined by the radiogram and rechromatography, o Final identification, however, is usually dependent on o the co-chromatography of the unknown, or questioned, radioactive material eluted from the paper with an authentic specimen of the suspected compound and the demonstration of the complete identi ty of the carrier material as determined by some visible test on the paper with the pat tern of radioactivi ty in the co-chromatogram. The amount of radioactivity incorporated in these compounds can be determined quite accurately by using the X-ray film as a means of defining that area of the paper containing the compound, thus per- mitt ing the particular spot to be cut out from the larger and eluted from the paper and mounted on a plate to be counted.

~NOkPYRUVATI~

PtIO IIP HOiIL~IT, E lq ATI[

iltIUI.OSr PHOlPi~iTI /~ ,~ . o s t ,ttOSPH,Tt " ~ I

~ T O I i [ PHOIPHATE I N~mOSC P H O ~ I ~ /

OkUe..Ollg ~ A T E ~l SI[D(MIPTUkO$I PHOSPHATI O

Itte~ut.otf m-H~IPNATI I HIXOII Dk~tIOIPHATI

IO life. Ptl II¢III l iPllt l t l

Fig. 5.--Radiogram of a paper chromatogram from 10 s C140~_ fixation in the light by Scenedesmus.

1 R. CONSDEN, A. H. GORDON, and A. J. P. ~IARTIN, Biochem. J. 98, 224 (1944). - A. A. BENSON, J. A. BASSHAM, M. CALVIN, T. C. GOODALE, V. A. HAAS, and W. STEPKA, J. Am. Chem. Soc. 7.?, 1710 (l:.)ao).

i t

/

/ t

t I

/ ?" PGb, t t

I I

t I

I /

O f t

/ , 'o

/ I I

i I

i I

/ e; /

/ O d,O~ Ohos~h~te j / , t ~ Mokc

)0 20 30 40 60 80 Time (Se¢,)

PSS

2 ° C

Fig. 6..-Behavior of radioactivity in specific compounds in extracts of Scenedesmus, exposed to radioactive carbon dioxide at 2°C.

A much simpler means would be to count the spot right on the paper with a GEIGER counter. The fraction of the total amount of radioactivity in the spot which is thus registered by the GEIGER counter is fairly constant for all compounds for any given chromato- graphic system. Thus, for most purposes it is sufficient simply to expose the paper to X-ray film in order to determine just where the radioactive spots are, and then having so defined them, to count them right on the paper for quanti tat ive comparison, by the GEIGER counter. I t is clear from Figure 5 tha t the var ie ty of products synthesized at room temperature by Scene- desmus (as well as by all other plants tried) is great, even in a very short time such as ten seconds. But even so, it is clear tha t the predominant compound as the time gets shorter is phosphoglyceric acid.

This is even more strongly demonstrated when the experiment is carried out at reduced temperature, for instance 2°C, so as to slow down all of the reactions and enable us to see more clearly the earliest products. Figure 6 shows a plot of the concentration of radio- act ivi ty per unit of algae for three of the major early compounds, formed at 2°C. On such a plot as this, it is clear that those substances which are formed directly from carbon dioxide with no appreciable intermediates

[15. XII. 1952] ~l, CALVIN and P. MASSINI: The Path of Carbon in Photosynthesis

Table I C xa Distribution in Photosynthet ic Products of Barley and Scenedesmus

449

Glyceric Acid Glycolic Acid Hexose

Conditions 1 --COOH --CHOH --CH2OH C 3,4 C 2,5 C 1,6

Barley P r e i l l u m :

2 m in d a r k . 4 s P S

15 s PS 15 s PS 30 s PS 30 s PS -~ 4 0 s P S 60 s P S

Scenedesmus 5 s P S

30 s P S 30 s PS~MI 7 . 60 s PS 6 60 s PS~MI 60 s PS ~ 60 s PS~MI

96 87 56 49

75

443

954

73 51 48

43

2"6 6"5

21 25

6

30

2"5

12 24 24

27

1"7 6"8

23 26

25

1.2

15 25 28

30

--COOH I ---CH~OH

48.5 51"5 50 ~_ 5 50 i 5

48 52

47 53

52

87 s

25 24

1 Experiments are steady-state photosynthesis, 10,000 footcandles unless otherwise stated. ~ 1000 footcandles. 3 Alanine obtained from this extract was 48O/o carboxyI-labeled. 4 Under the same conditions, Chlorella produced phosphoglycerate lateled 98%, 3% and 2%, respectively, n In this extract, malic acid was labeled 6.5% and aspartic acid 4% in the non-carboxyl carbons. ~ 3000 footcandles.

" Malonate inhibited.

lying between them and carbon dioxide will be the only ones that will show a finite slope; all others should start with a zero slope. A finite slope is certainly the case for phosphoglyceric acid and possibly for malic acid, in- dicating at least t~vo independent carbon dioxide fixing reactions, one leading to a three-carbon compound and the other producing a four-carbon compound s.

Since the hexose phosphates appear extremely early in all of these photosynthesis experiments and because of the known close relationship between the hexose phosphates and phosphoglyceric acids in the glycolytic sequence, it seemed most reasonable to suppose that these hexose phosphates were formed from the phosphoglyceric acid by a combination of the two three- carbon fragments derived from phosphoglyceric acid in an overall process very similar to, if not identical with, the reversal of glycolysis.

One means of testing this suggestion would be a comparison of the distribution of radioactivity in the three carbon atoms of glyceric acid with those in the hexose as shown in Table I. I t thus appears tha t the hexose is indeed formed by the combination of two three-carbon molecules derived from the glyceric acid in such a manner that carbon atoms three and four of the hexose correspond to the carboxyl-carbon of the glyceric acid; carbon atoms two and five with the alpha-carbon; and carbon atoms one and six with the beta-carbon of the

1 E. J. BADIN and M. CALVIN, J, Am. Chem. Soc. 72, 5266 (1950). - S. KAWAGUCltl, A. A. BENSON, M. CALVIN, and P. M. HAYES, J .Am. Chem. Soc. 74, 4477 (1952).

glyceric acid. This correspondence is maintained when the distribution in these two compounds (glyceric acid and hexose) is compared for a wide variety of different times.

With this clear cut indication of the similarity between the path of hexose synthesis and the known path of its breakdown, another means of testing how closely this parallelism might be followed suggests itself. The hexose derivative which is last in the sequence of changes prior to the breakdown of the carbon skeleton during glycolysis is the fructose-l,6-diphosphate. Correspondingly, then, it presumably would be the first hexose derivative to appear in the reverse direction. If this is the case and, furthermore, if the hexose derivative reservoirs involved in sucrose synthesis are more or less isolated from those involved in storage and glycolysis, the radioactivity should appear in the fructose half of the sucrose molecule prior to its appearance in the glucose half. This is indeed the casO. However, sucrose does not seem to be formed by the simple reversal of the sucrose phosphorylase system which was described for certain bacteria *, since for this to be the case, free fructose would have to be apparent in the photosynthesizing organism, whereas it is never so found, nor has the enzyme itself ever been isolated from any green plant.

x S. KAWAOUCm, A. A. BENSON, N. CALWS, and P. M. HAYES, J. Am. Chem. Soc. 74, 4477 (1952).

W. Z. HASSID, M. DOUDOROFF, and H. A. BARKER, J. Am.Chem. Soc. 66, 1416 {1944). - M. DOUDOROFF, H. A. BARKER, and W. Z. HASSlD, J. Biol. Chem. 16,% 7o5 (1947).

Exper, 29

450 M. CALVIN and t ). MASSXNI: The Path of Carbon in Photosynthesis [EXPERIENTIAVoL.VIII/12]

The recent identification I as uridine diphospho- glucose (U.D.P.G.) of the spot which had been previously 2 called , the unknown gIucose phosphate spot , has lead to another suggestion as to the mode of formation of sucrose. Glucose-labeled U.D.P.G. appears very early in the sequence of compounds formed. Fur- thermore, it has been possible to demonstrate the presence in the hexose monophosphate area of a sucrose phosphate by using a carefully selected phosphatase, containing no invertase, in the treatment of this entire phosphate area 3. We have suggested, therefore, that U.D.P.G. may be involved in sucrose synthesis in a manner similar to that of glucose-l-phosphate in the numerous phosphorylase reactions, with the difference, however, that the acceptor of the glucose moiety would be some phosphate of fructose, thus producing a sucrose phosphate. Recent work by PUTNAM and HASSlD 4 gives further support to the idea that only phosphorylated derivatives of glucose and fructose are involved in sucrose synthesis in higher plants. They found that in sucrose synthesis, from labeled glucose in leaf punches, no free fructose was formed, although the sucrose becomes equally labeled in both the glucose and fructose portions. Conversely, when labeled fructose is used, no free labeled glucose appears, while the sucrose is uni- formly labeled in both moieties.

I t is possible that compounds of the U.D.P.G. type could be concerned in the transformation of sugars and the subsequent incorporation into polysaccharides. Uridine diphosphate would thus serve as a carbon

• carrier in the same way that pyridine nucleotides and flavonucleotides are involved in hydrogen transfer; the adenylic acid system in phosphate transfer; and coenzyme A in the transfer of acetyl groups. There is already some evidence for the existence of other members of the uridine diphosphate group from our own work, as well as tha t of others 5.

We may now turn our attention from the fate of the glyceric acid to the problem of its origin. An examination of TaMe I indicates quite clearly that the first position in the glyceric acid to become labeled is the carboxyl group. As time proceeds, the other two carbon atoms in the glyceric acid acquire radioactivity and it

1 j . G. BUCHANAN et al., in press. - J. G. BUCHANAN, J. A. BASS- HAM, A. A. BENSON, D. F. BRADLEY, }I. CALVIN, L. L. DAUS, M. GOODMAN, P. M. HAYES, V. H. LYNCH, L.-T. NORRIS, and A. T. WILSON, Phosphorus MeMbolism, Vol. II (Johns Hopkins Press, Baltimore, Maryland, 1952), in press.

2 S. KAWAGUCltI, A.A. BENSON~ N. CALVIN~ and P. M. HAYES, J. Am. Chem. Soc. 74, 4477 (1952).

a j . G. BUCHANAN, J. A. BASSHAM, A. A. BENSON, D.F. BRADLEY, M. CALVINt L. L. DAYS, M. GOODMAN, P. M. HAYES, V. H. LYNCtf, L. T. NORRIS, and A. T. WILSON, Phosphorus 3Ietabolism, Vol. I I (Johns Hopkins Press, Baltimore, Maryland, 1952), in press. - J. G. BUCHANAN, in press.

¢ E. W. PUTNAM, Thesis (University of California, Berkeley, 1952). 5 R. CAPUTT0, L. F. LELOIR, C. ]~. CARDINI, and A. C. PALADINI.

J. Biol. Chem. 184, 333 (1950). - A. C. PALADINI and L. F. LELOm, Bioehem. J. 51, 426 (1951). - J. T. PARK, J. Biol. Chem. 19J, 885 (1952).

appears tha t they acquire it at equal rates, at least within the present accuracy of the experiments.

I t thus appears that the most rapid reaction which carbon dioxide can undergo at least at high light in- tensities, is a condensation with a C 2 fragment leading directly to phosphoglyceric acid. An examination of the chromatograms of a very short photosynthetic period shows glycine and glycolic acid as the only two-carbon compounds present. The distribution of radioactivity among the carbon atoms of these two compounds is always equal and the same and corresponds very well with that in the alpha- and beta-carbon atoms of the glycerie acid, as may be seen from Table I. This suggests tha t glycolic acid either is in the direct line for the formation of the C 2 carbon dioxide acceptor, or is very closely related thereto.

The question now arises as to the source of this C 2 carbon dioxide acceptor. There are, of course, only two possibilities for its origin. Either it results from a one- plus-one combination or it must result from the split- ting of a four-carbon compound or a larger one. In order for it to result from the combination of two one-carbon fragments there must exist as an intermediate some one-carbon compound more reduced than carbon dioxide which, in turn, may combine either with itself or with carbon dioxide. Furthermore, the reservoir of this one-carbon intermediate would have to be vanish- hagly small since all a t tempts to find labeled, reduced, one-carbon compounds, such as formic acid or formal- dehyde, in the early stages of photosynthesis have failed and, in addition, the resulting.two-carbon fragment is very nearly equally labeled in both carbon atoms.

One would also expect that these one-carbon compounds would tend to disappear under conditions of low carbon dioxide concentrations leading to the disap- pearance of the two-carbon condensation product resulting from them. This leads Us to the supposition that the formation of glycolic acid would be expected to drop off under conditions of low carbon dioxide concentration which is the reverse of what is observed.

We are thus left with the following possibility for the C 2 compound--the cleavage of some C a or larger struc- ture. The fact of the early appearance of label in malic acid, taken together with the lack of any appreciable amounts of label in the compounds of the tricarboxylic acid cycle 1, led us to the supposition that malic acid was either a precursor to, or very closely related to, a four-carbon compound which could be split to produce the required two-carbon fragment.

In the course of the search for the two-carbon acceptor, and its immediate precursors, two new compounds were identified as early products of carbon dioxide incorporation which seem to have little to do with the direct synthesis of hexoses and, therefore, had a very likely function in the regeneration of the two-

1 A. A. BENSON and M. CALVIN, J, Exptl. Botany 1, 63 (1950).

[15. XII . 1955] M. CALVIN and P. MASSI,~I : Tile Path of Carbon in Photos~qithcsis 451

carbon acceptor. These were the phosphates of the seven carbon sugar sedoheptulose and of the five carbon sugars ribulose, ribose and arabinose 1.

The question immediately presents itself as to the relation between these two compounds along the path of carbon assimilation, not only with each other but with the precursors which are already known and the possible products that might be formed from them. The at tempt to answer this question focuses our attention once again upon some of the shortcomings and limitations of the method of observation that we are using and the nature of the experiment which we are performing. Our initial hope of determining the .sequence of intermediates by a simple observation of a sequence of compounds into which radioactivity has been incorporated in steady state experiments is now complicated by the uncertainty as to the amount of compound present during the steady state. It is easy to visualize a situation in which the actual amount of intermediate present during the steady state is so small as to escape observation by our methods, or perhaps even to be so unstable as to be lost by our methods of observation. This complete failure of a compound to appear on a chromatogra m, although it might con- ceivably be an intermediate, is, of course, an extreme case. The more usual situation is one in which most of the intermediates are present but in varying concentrations in the steady state. Under such conditions a single or even several observations of the relative amount of radioactivity incorporated into a variety of compounds would not necessarily be any real criterion of the relative order of these compounds in the sequence of events.

In order to achieve the full value of the method of observation then, it becomes necessary to perform rather extended kinetic experiments in which the appearance of radioactivity in all compounds is plotted as a function of time at sufficiently short intervals to enable a rather accurate and detailed curve to be obtained. Furthermore, the distribution of radioactivi ty among the atoms within each compound should also be determined as a function of time. The validity of any proposed sequence of events could then be determined by a comparison of the calculated appearance and distribution curves with those actually observed. In order to calculate such appearance curves, as well as the distribution curves amongst the atoms in each compound, one can set up a system of linear differen- tim equations based upon the following model:

R 12 R C02 • A • B ~ - - - ~ ' S (1)

where C02 represents the entering carbon dioxide; A, B, etc. represent intermediates involved in carbon

x A. A. BENSO~, J. A. BASSItAM, M. CALVIN, A. G. HALL, H. E. H1RSCH, S. KA~VAGUCHI, V. H. LYNCH, and N. E. TOLI3ERT, J, Biol. Chem. 196, 703 (1952).

dioxide assimilation; S represents more or less final storage product; R is a measure of the total rate of carbon dioxide assimilation in the steady state expressed in moles of carbon per minute.

The rate of change of the specific activity of a single carbon atom in A, given by XA, is then expressed by Equation (2). (The specific activity of the entering carbon dioxide is here taken as unity. [,4], the concentration of the compound A, is independent of time.)

d X A _ R ( l - X 2 . (2) dt EA;

The specific activity of the corresponding atom in compound B is given by an exactly similar Equation (3).

dXB R dt EB~ ( X ~ - Z . ) . (3)

Equations of identical form may be written for every atom of every compound that might be considered an intermediate. These equations may be solved expli- citly by means of a differential analyzer provided two parameters are known. These are the total rate of entry of carbon into the system during the steady state, R, and the steady state concentration of each atom which might be considered as lying along the path of carbon assimilation [A], [B], e tc .

I t is clear that if such compounds (whose prime function it is to serve as carbon carriers between the entering carbon dioxide and the final storage products in the plant) do indeed exist in biological systems they would very soon become saturated with radioactivity. By this is meant that the amount of radioactivity observed in that particular compound would very soon reach a maximum value and remain that way. The reason for this is that by definition the amount of these intermediate compounds is not changing, and also is small compared to the total amount of carbon the plant assimilates during the experiment. Since all of the carbon, or at least most of it, must pass through these reservoirs of intermediates "they will very soon acquire the same specific activity as the entering carbon dioxide. In contrast to this, those materials which are not functioning as simple intermediates but rather are functioning as storage reservoirs, or are very distant from the immediate photosynthetic intermediates, will not acquire radioactivity as rapidly, or if they do they wilt not become saturated as rapidly as those which are directly involved in the path of carbon assimilation. The amount of radioactivity found in those compounds which saturate in a relatively short time now provides a relatively easy method of determining the size of the functioning reservoirs of these compounds which are directly engaged in the path of carbon assimilation. A simple measurement of this amount compared to the specific activity of the entering carbon dioxide will provide a measure, in moles per unit volume of the

29*

452 .~t. C.&LViN and P. MASSINI: The Path of Carbon in Photosynthesis I EXPI,.'RII~NTIAVOL.VIII/I'2]

biological material, of the compound in question. Furthermore, having once achieved a relatively uniform label in these photosynthetic intermediates, it becomes possible to follow the behavior of the reservoir size as a function of change in external variables, for example, light intensity. We have chosen to include in this review a more or less detailed description of just this determination of the effect of light intensity upon reservoir sizes as a means of describing the general experimental technique which is involved.

Steady state and reservoir sizes--Methods and results

The apparatus used for these experiments was con- structed to permit the algal suspension to be left under controlled external conditions (illumination intensity, temperature, carbon dioxide and oxygen concentration) while photosynthesizing for at least one hour. Further- more, it was required that the change, natural to radioactive carbon dioxide, which was to be circulated in a closed system, and the withdrawal of several samples at given time intervals be accomplished with a mini- mum of change in these conditions.

The apparatus consisted of: (a) A square illumination vessel A (Fig. 7) made out

of Lucite (polyacrylic plastic), 49 cm high, 11 cm wide and 0.7 cm thick (inside dimensions). The bottom was provided with a gas inlet tube with five small holes to allow good contact between gas and liquid and a drain tube closed with a screw clamp. The top of the vessel was provided with a gas outlet tube. A water-alcohol mixture from a constant temperature bath was allowed to flow over the outer surfaces of the vessel in order to control the temperature of the suspension.

f

E

I%

Fig. 7.-~r)iagram of the assembly for stead)" state photosynthesis. (For explanation of the letters, see text,)

(b) Two illumination banks (represented by B), each with four fluorescent tubes (General Electric, quality white, 20 W each), providing an almost uniform illumination over the whole surface of the vessel, of 7 × 104 ergs./cm z (roughly 700 footcandles).

Fig. S.--Assembly for s teady state photosynthesis. (For explanation of the letters, see text.)

(c) An ionization chamber C, connected to a record- ing vibrating reed electrometer, to record the activity of the gas leaving the vessel continually during the run.

(d) Three gas traps D, to permit the addition of a known amount of radioactive carbon dioxide to the system, and trap the remaining radioactivity after the run.

(e) A flask E, of 5 1 volume, containing a mixture of 1% radioactive carbon dioxide in air. The reservoir contained so much carbon dioxide that the algae assi- milated no more than 20% of it during a run.

(]) A gas circulating pump F of the rubber tubing type, and a flow meter G.

(g) A system of four-way stopcocks H, which per- mitted the vessel to be flushed with a mixture of 1% ordinary carbon dioxide in air, from the cylinder I. The assembly is shown in Figure 8.

In a typical experiment, 2 cm 3 (wet packed) of one- day old Scenedesmus, washed and resuspended in 200 cm "~ of deionized water, were placed in the vessel and aerated with the ordinary gas mixture for at least one- half hour, while the mixture of radioactive carbon dioxide circulated in the gas system for thorough mixing, without passing through the vessel. The suspension was kept at 24 °C. After this time, during which a steady state of photosynthesis had been reached, the radioactive mixture was passed through the vessel in place of the ordinary gas mixture, by a manipulation

! 13. XI I . 1:t52.I M. CALVL~ a n d 1 ), .MAss),~I: T h e l'ath of C a r b o u iu l ' h o t o s y n t h t ' s i s 453

O

*~ ~ ~ ~4ENEOEM

D

Fig. 9.--Radiogram of a paper chromatogram from 10 min, C1402 fixation in light by Scenedesmus, 1 % suspens ion , 1 % CO~ in air; light intensity 7 x 104 ergs. /cmZ-s. D , H . A . P . ; dihydroxyacetone phosphate ; P.E.B.:phosphoenolpyruvic acid; P.M.P.:pentose monophosphates; P. Go.A, :phosphogtyeolic acid; P.G.A. :phosphoglyceric acid; H . M . P . :

hexose m o n o p h o s p h a t e s ; D . P . : p e n t o s e a n d hexose diphosphates.

of the pair of stopcocks at H, and samples of 20 cm s of the suspension withdrawn at intervals of five or ten minutes. These samples were dropped into 80 cm 3 of alcohol of room temperature, to make an extraction in 80% alcohol. After 30 min of photosynthesis, the

lights were turned off and the suspension allowed to remain in the dark for a period of 5 min, during which time again several samples were withdrawn, and treated in the same manner. In one experiment another light period followed the dark period.

The samples were shaken for 1 h and centrifuged. The residue was re-extracted in 50 cm 3 of 205/o alcohol at room temperature, centrifuged, and re-extracted again with 20 cm 3 of water. The extracts were concen- trated together to 0.5 cm 3.

An aliquot of the concentrate equivalent to 30 y1 of packed cells was evaporated on a corner of a filter paper (WHAT~AN # 1), and the chromatogram run with water- saturated phenol in one direction and n-butanol- propionic acid-water in the other. The chromatograms were exposed to X-ray film for about two weeks/. The labeled compounds appeared on it as black spots. Figure 9 shows the radiogram for ten minute photosynthesis of Scenedesmus. The amount of radioactivity contained in the different compounds was determined by counting the corresponding spots on the paper directly with a large-area GEIGER-MLTLLER tube with thin mica window. The compounds were identified by a combination of the following criteria: (a) Their position on the paper; (b) the spot was cut out, eluted from the paper with water and run again in suitable solvents, together with such an amount of the suspected corn-

50

< . . . . . . . . . . . . . . . LIG ~ x

\

\ \ \

\ \ \

\

LIGHT: ~ ,~ IO 2"0 3 DARK: 0 2 5

TIME (rain.) OF EXPOSURE TO Gt40Z

4O

o O x (t) .J .J ,~ so 6 o et to t~

z

Z

0 0

F ig , 10.--C1402 fixation by Scenedesraus. 1 % suspens ion , I OZ/o CO~ in air, light intensity 7 x 104 ergs.]cm2-s.

& . . . . -~ DIPHOSPHATES

tS~- 0 ....... 0 PHOSPHOGLYCERIC ACID I ~, I o---o MONOPHOSPHATES (HMP AREA) ] . - - . SOCROS / ,

~ D - - - - - - ~ - - O I t O- ........... "v"'=*,~ ¢ ' ~

DARK: TIME (ram.) OF EXPOSURE TO 0402

2 5

Fig. l l . = - B e h a v i o r of radioactivity in specific compounds in the ex- t r a c t from the experiment of Figure 10.

pound that it could be detected by a specific spraying reagent. The black spot on the film had to coincide accurately with the color reaction; (c) the eluted spot was chemically transformed (e.g. treating the sugar phosphates with phosphatase) and the resulting compound cochromatographed with carrier detectable by spray.

Figure 10 shows the total and the extracted amounts of radiocarbon fixed by 1 cm 3 cells during 30 min of

1 hi. CALVIN, J . Chem. E d u c a t i o n 26, 639 (1949).

454 M. CALVIN and P. MASSINX: The Path of Carbon in Photosynthesis [EXPERIr:NTIAVoL.VIII]12]

\

I'= e--e SUCROSE ~ k ~] / ~ - - ' ~ MALIG ACID ~ k l o--o OLU,A.,O *c,o / I!,]'

o 13--{3 c'rR'° A°'° / i ~

LtGHX~ LI DARK: , I ,?. ,$"

T;ME (m~,) OF I[XPOSURE TO GI403

~,~'~IGHT *

\

\ \

Fig. 12.--Behavior of radioactivity in specific compounds in the extract from an experiment done under conditions corresponding to

those of Figure 10.

photosynthesis followed by 5 rain of darkness. The slope in the total fixation curve in the light corresponds to a 13 cm 3 CO s assimilation (N.T.P.) per hour.

Figure 11 shows the amount of radioactivity incorporated into sucrose and three phosphorus compounds for the experiment of Figure 10.

Figure 12 gives the number of counts in sucrose, glutamic, malic and citric acid, for a different experiment of 15 min photosynthesis, followed by 10 rain dark, and again 5 min of photosynthesis.

Although the variation between experiments is quite high, there are some striking features which are com- mon to all:

(1) The curves of some of the compounds show a marked decrease in slope after 5 min of photosynthesis. This quite clearly indicates the presence of rapidly turning-over reservoirs in the photosynthesis cycle which are then thoroughly labeled and reach the specific activity of the fed carbon dioxide : Diphosphate area (mainly ribulose diphosphate) ; hexose-monophosphate area (50% glucose-, 26% sedoheptulose-, some fructose- and mannose-monophosphate); phosphoglyceric acid. The leveling off of these curves permits the calculation of the concentration of the reservoirs of those compounds in the photosynthesis cycle, by divid- ing the measured amount of radioactivity per carbon atom by the specific activity of the fed carbon dioxide 1.

Table II gives the steady state concentrations during photosynthesis for some compounds determined by this method.

(2) The fact that the activity vs. time curves show a definite yet low slope for as long as 30 min can be taken to indicate that the breakdown of carbohydrates

l~b~ I I

Steady State Concen~at ionsofSome Compounds Involved in the Photosynthesis Cycle. Scenedesmus, experimental conditions as in

Figure 10

Substance /tmoles/cm ~ cells t

Phosphoglycer ic acid Dihydroxyace tone phospha t e Fructose phospha te . Glucose phospha t e Mannose phospha t e . Sedoheptulose phospha te Ribulose d iphospha te Alanine

1.4 0.17 0.12 0.4 0.05 0.18 0.5 0.2

continues throughout the illumination, i,e. their for- marion from photosynthetic intermediates is revers- ible. Thus, there are two sources of the intermediates.." (a) the carbon dioxide fed; the amount of compound formed from this source reaches the maximum specific activity in 5 to 10 min; (b) the carbohydrate pool of the cells; the amount formed from this source is labeled only slowly since the specific activity of the carbohydrate pool rises slowly due to the large size of the pool.

(3) Other compounds show almost constant rate of labeling during the whole period of photosynthesis; sucrose, malic and glutamic acid. For this and other reasons it is clear that these compounds are not in the photosynthesis cycle, but are formed during the photosynthesis at a constant rate. Their large reservoirs in the cells are labeled only slowly.

Table I I I

Phosphatase Treatment of H.M.P. Area after 30 rain Photosynthesis and 30 min Photosynthesis Followed by 5 min Dark

Substance

Glucose F ruc tose Sedoheptulose Mannose

Number of counts/min on paper

30 min P.S. 30 rain P.S. 5 rain D.

3140 4280 910 1040

1600 } 460 12103

(4) When illumination is interrupted there appears a sudden great increase in the concentration of phosphoglyceric acid (followed by a slow decrease after 2 rain), and an almost complete depletion of the diphosphate area. Analysis of the monophosphate area showed that the amount of sedoheptulose phosphate decreased also (cf. Table III). The concentration of

1 The efficiency factor of the counting of spots on papers has been determined by converting three cut out spots to barium carbonate and measuring their activity in an ionization chamber. I t is 19 disintegrations per count.

1 Volume measured as wet packed cells An appreciable fraction of this count is certainly hexose so that

one may estimate a maximum value of the heptose at around 800 counts/rain.

[15. X I I . 1952] M. CALVIN and P. MassINl : The P a t h of Carbon in Pho tosyn thes i s 455

malic acid decreases as well. The rate of labeling of glutamic acid is increased greatly after a short induc- tion period; citric acid, which contains little activity during the whole light period, shows a sudden increase in the dark, followed by a slow decrease. The labeling of sucrose continues at the same rate as in light for about 1 rain, after which it is stopped almost com- pletely.

Both experiments gave the same picture for most of the compounds, with the two exceptions : In the second experiment the diphosphate area, which in the first contained almost the same number of counts as phosphoglyceric acid during the light, had only about 15°/o of it in this second run. This value dropped to 5% in the dark. The phosphoglyceric acid showed a hardly significant rise in the dark during the first 2 min, but again a slow decrease after 5 min. Although we do not know why in this experiment the concentration of ribulose diphosphate was so low in the light, the co- incidence with the lack of increase of phosphoglyceric acid points to a connection between both effects.

(5) In the light following the dark, the diphosphates, phosphoglyceric and malic acid increase again.

60sL • 1.8 GLUTAMIC

60sL-6OsO 37 ACID

120sL ~ I0

60sL • 2.6 CITRIC

60sL-6OsO 43 ACID

120sL ~ 8.2

60sL ~ I69

60sL-6OsD 327

120sL 373

ALANINE

60sL ~ 9l SUCROSE

60sL-6OsO ~ 173

t2OsL 526

Fig. 13. - -Effect of l i g h t and d a r k on the labe l ing of g l u t a m i c and c i t r ic acid. 0-1% suspension, l i g h t i n t e n s i t y 1.6 x l 0 s ergs]cm~-s (Numbers :

counts / ra in x 10 -~ on pape r pe r cm a cells).

The effect of dark on the labeling of glutamic and citric acid was already reported in an earlier paper 1 and studied more closely in the following experiments: 0.2 cm a wet packed algae (Chlorella pyrenoidosa) were suspended in 200 cm 8 distilled water, illuminated in a flat circular vessel of 1 cm thickness by incandescent lights through an infrared filter (intensity 1.6 × 105ergs/ cm2-s) and aerated with 0.08% carbon dioxide in air. The low concentration of cells was chosen to avoid shad- ing of cells in the suspension, so that during the light period all the cells were illuminated continually.

I M. CALVlS, J. Chem. E d u c a t i o n 96, 639 (1949),

After one-half hour, the aeration bubbler was taken out and a suitable amount of radioactive bicarbonate (sodium) solution added. (The algae, which were grown in slightly acid medium, had enough buffering capacity to convert the bicarbonate to carbon dioxide). The vessel was immediately stoppered and shaken in the light. After 1 rain, the suspension was drained into

CARBOHYDRAT~$ PROTEIr;~

FATS

~v Fig. 1,1.

a darkened flask, and after another minute poured into four times its volume of boiling alcohol. Control samples were treated in the same way, but kept in the light, in contact with radioactive carbon dioxide for 1 and 2 mill, respectively. The analysis of the fixed radioacti- v i ty was performed by paper chromatography and radioautography with the technique already described. The results are shown in Figure 13.

Discussion

It has already been pointed out that photosynthesis is not a mere reversal of respiration; this was supported by the observation that the carbon of newly formed photosynthetic intermediates is not available for respiration while the light is on 1. VVe may thus represent the relationship between photosynthesis and respiration by the following scheme (See Figure 14). The labeling of the KREBS cycle intermediates through the storage products (carbohydrates, fats, proteins) of the cells is a slow process, due to the relatively large size of the storage pools. The fact that the photosynthesis intermediates find their way into the tricarboxylic acid cycle very rapidly after the light is switched off means that there is another connection between the two cycles which is blocked as long as the light is on but becomes accessible in the dark. This was interpreted in earlier work z in terms of the action of the light in maintaining at low concentration the intermediate required for entry into the tricarboxylic acid cycle. A closer specification of how this is accomplished is now possible since the discovery that alpha-lipoic acid is a

1 M. CALVIN, J. Chcnx. E d u c a t i o n 26, 639 (1949). - J . W. WgmL, P. ~I. WARRINGTON, and M. CALVIN, J, Am. Chem. Soc. 73, 5058 (1951).

2 ~[. CALVIN, J. Chem, E d u c a t i o n ,26, 639 (1949).

456 M CALVIN and P. MASSINI: The Path of Carbon in Photosynthesis [EXI'ERIENTIAVoL.VIII/12]

cofactor for the oxidative decarboxylation of pyruvic acid to an active acetyl group x which is the one reaction known to feed the KREBS cycle z. The mechanism of the reaction may be written this way:

CH~ / \

CH 2 H C - CH2--CHz-A2H2--CO--Thiamin+ CHa--CO--COOH ! i (Co-pyruvate oxidase) (Pyruvic acid)

S --S

CH2 / \

CH~ CH - 12

I I s s

/ J CH3--CO COOH

Coenzyme A >

CH~ / \

CH 2 C H - R + Acetyl CoA+ CO 2

t .I S S

1 I H H

The reduced lipoic acid complex would then be reoxi- dized to the disulfide form by a suitable oxidant (e.g. pyridine or flavin nucleotides). In order that the oxidation of pyruvic acid can proceed, the enzyme has to be present in its oxidized form. tf it is kept in its reduced form under the influence of the light-produced reducing power, the reaction cannot proceed and the pyruvie acid formed during photosynthesis will not find its way into the respiratory cycle. The reaction is inhibited because only a small amount of the enzyme catalyzing it exists in the required form, most of it being kept in the other form under the "pressure" of the reducing power generated by the light energy. This recalls a similar phenomenon which has been known for a long time, i.e. the suppression of the fermentation of carbohydrates in favor of their oxidation under aerobic conditions (PASTEUR effect). This effect has been explained in a manner similar to the one used here to account for the inhibition of the respiration of photosynthetic intermediates 3. The reduction of acetaldehyde to alcohol requires a dehydrogenase in its reduced form; under aerobic conditions the dehydrogenase exists primarily in its oxidized form, and the acetaldehyde instead of being reduced is oxidized to acetic acid.

The sudden rise in phosphoglycerie acid and the decrease in ribulose diphosphate aiad sedoheptulose

phosphate in the dark period, together with the observation that the dark rise in phosphoglyceric acid is absent when the ribulose diphosphate concentration was low during the light, confirms the earlier suggestion that the phosphates of the C 7 and C~ sugars are precursors of the C2 carbon dioxide acceptorL This, together with evidence gathered in previous work ~ leads to the following scheme for the photosynthetic cycle 3 (Fig. 15).

Upon this basis an at tempt might be made to relate the two effects as follows; ,nhen the light is turned off, the reduction reactions requiring light are stopped, whereas cleavage and carboxylation reactions continue until their substrates are exhausted. Presumably, this would lead to a depletion of the C~ and C 7 sugars, the synthesis of which requires reduction steps (particularly the six-equivalents leading to the tetrose which itself is a very small reservoir), and a rise of phosphoglyceric acid, the further fate of which is also dependent upon reduction. However, a number of arguments seem to contradict this view: (1) The observation that plants fix radiocarbon in the dark immediately following a light period at low carbon dioxide concentration, to form a similar pattern of compounds as the one found in photosynthesis shows that the sequence following phosphoglyceric acid is not Mocked at once upon cessation of illumination, but that the cells contain sufficient reducing power to transform some phosphoglyceric acid into carbohydrates; (2) the cleavage of the pentoses and heptoses into the C 2 carbon dioxide acceptor and a triose and pentose respectively is dependent on a reduction step as well.

co a z [H/ ca ~C3 . . . . . . . ~ ~ , C 4

zN'eNrI~se, r.~se .E:xose ~,/ - - %

H E P T O S E T E T e . 3 S E "

Fig. 15.

*C4 MALIG AGtO

We are thus led to the suggestion that the rise in phosphoglyceric acid is not be explained by a mere interruption of the sequence, but that the rate of pro- duction of phosphoglyceric acid at some time in the

1 L. J. REED, I. C. GUNSALUS, etal., J. Am. Chem. Soc. Z3, 5920 (1951). - E. L. PATTERSON, et aI., J. Am. Chem. Soc. 7J, 5919 (1951). - I. C. GVNSALUS, L. STRUGLIA, and D. J. O'KANE, J. Biol. Chem. 19t, 859 (1952). - L. J. REED and B. G. DEBUSK, J. Am. Chem. Soc. 7g, 3457 (1952). - M. W. BULLOCK, et al., J. Am. Chem. Soc. 74, 3455 (19~2).

S. OcHoA, J. R. STERN, and M. C. SCHNEm~R, J. Biol. Chem. 193, 691 (1951). - S. KORKES, A. D~L CAmLLO, L C. GUNSALUS, and S. OCHOA, J. Biol. Chem. 193, 721 (1951).

O. MEYERHOF, Amer. Scientist 40, 483 (1952).

A. A. BENSOS, J. A. BASS~A*L M, CALW~, A. G. HALL, H..E. HIRSCH, S. KAWAGUCHI, V. H. LYNCII, and N. E. TOLBERT, J. Biol. Chem. 196,708 (1952).

2 S. KAWAGUCItI, A. A. BENSON~ hi. CALVIN, and P. M. HAYES, J. Am. Chem. Soc. 74, 4477 (1952). - M. CALVIN, The Harvey Lectures t6., ~13-251, 1951, in press.

This scheme is intended to represent 01113, changes in the carbon skeletons. The reducing equivalents are indicated only to show redox relationships between the known compounds. A number of the isolated compounds are isoximers and have not been included.

[15. XII. 1952] M. ChLvln and P. ~[ASSINI: The Path of Carbon in Photosynthesis 457

HOsP--OCH~--CHOH--CHOH--CO--CH2--O--P03H

CO 2, 2[H]

CO~

dark

-HO3P--OCHz--CHOH--CHO phosphoglyceraldehyde

-HOsP--OCH2--CHOH--COOH phosphoglyceric acid

2x-HOsP--OCHa--CHOH--COOH phosphoglyceric acid

first minute of darkness is actually higher than it is in the steady state photosynthesis. This would be the case if the Ca-C 2 cleavage of ribulose diphosphate, which in photosynthesis presumably yields a triose phosphate molecule beside the C 2 carbon dioxide acceptor, in the dark yelds a molecule of phosphoglyceric acid instead of the triose molecule. The overall reactions may be represented above (not a mechanism).

This hypothesis is supported by the fact that the triose phosphate also decreases in the dark.

The fact that the net result of the reaction sequence in the light from ribulose diphosphate to phosphoglyceric acid and triose phosphate is a reductive carboxylation and thus the reversal of the oxidative decarboxylation which, in the case of pyruvic acid, requires the presence of a cyclic disulfide compound leads to the idea that the former sequence might be catalyzed by a similar enzyme. This idea seems to be supported by the results of an experiment performed in this laboratory some time ago, which were difficult to explain 1.

In order to examine the relation between photosynthesis and the glycolytic cycle, a series of experiments similar to those described previously were performed with added iodoacetamide which is known to inhibit the action of triose phosphate dehydrogenase ~, presumably through a reaction with its sulfhydryl group s. A 1% suspension of Chlorella in phosphate buffer was allowed to photosynthesize in light of 2500 footcandles and an atmosphere of 1% carbon dioxide, 5% oxygen and 94% nitrogen. At various times before adding the radioactive bicarbonate solution, iodoacetamide was added to give a 1.5 × 10 -~ M solution. 1 min after adding the radiocarbon, the cells were killed and extracted.

After 8 rain contact with iodoacetamide, the cells were still able to fix 75% as much carbon dioxide as non-poisoned cells otherwise treated the same way (control). The amount of radioactivity in phosphoglyceric acid was 50% of the control, and the amount in sucrose had reached a sharp maximum of 3.5 times

1 W. STEPKA, Thesis Universi ty of California (June 1951). 2 0 . MEYERIIOF and W, KIESSLING, Biochem. Z. 281, 249 (1953). 3 L. RAPKINS, C. r. Soc. Biol, (Paris) 11"2, 1294 (1933).

that in the control. There was practically no radio- act ivi ty in the ribulose diphosphate. After 90 min of exposure to the poison the cells had practically lost, their ability of photosynthesis.

If, in the proposed photosynthetic cycle, the cleavage of the heptose and pentose phosphates is dependent on an enzyme containing sulfhydryl groups, which were more sensitive to iodoacetamide than the triose phosphate dehydrogenase, a picture similar to the one described would be expected: After short exposure to the poison, in relatively low concentration, the lack of C a carbon dioxide acceptor would slow down the photosynthetic cycle. The synthesis of carbohydrates, however, would proceed almost without inhibition, thus decreasing the concentrations of the intermediates in the cycle. This would allow the compounds to reach a higher specific activity during the period of exposure to radiocarbon (cf. equation (2), change of specific activity inversly proportional to concentration]. At some time after administration of the poison, the sucrose would be labeled faster than in the control due to the higher specific activity of its precursors. After a longer period, however, the rate of synthesis of sucrose would decrease because the pool of its precursors would be exhausted.

Zusammen/assung

Die Trennung des Ph~nomens der Photosynthese griiner Pflanzen in eine Lichtreaktion und die vom Licht unabhiingige Reduktion der Kohlens~ure werden dis- kutiert.

Die Reduktion der Kohlens~ure und das Schicksal des assimilierten Kohlenstoffs wurden untersucht mit Hilfe der Spurenmethode (Markierung der assimilierten Koh- lens~ure mit C 14) und der Papierchromatographie. Ein Reaktionszyldus wird vorgeschlagen, in dem Phosphogly- zerins~ure das erste isolierbare AssimiIationsprodukt ist.

Analysierung des Extraktes von Algen, die in einem station~ren Zustand fiir l~ngere Zeit radioaktive Kohlen- siiure assimilierten, lieferte weitere Auskunft fiber den vorgeschlagenen Zyklus und gestattete, die am Zyklus beteiligten Mengen einiger Substanzen ungef~hr zu be- stimmen. Die friihere Vermutung, dass Licht den Res- pirationszyklus beeinflusst, wird best~tigt. Die M6glich- keit der Mitwirkung von ~-Liponsiiure (~-lipoic acid) oder einer verwandten Substanz, bei diesem Effekt und im Photosynthesezyklus, wird erSrtert.

Documents

EXPERIENTIA - USP · 2015-03-17 · plant is, in its first phase, a straightforward one. C02 , 1 ~o2 h~ l LEAF T H~ % T (CH20) Fig. 2. We may visualize the problem in terms of the